T-box3基因对结肠癌细胞增殖、凋亡的影响及其机制

Effect of T-box3 gene gene on proliferation,apoptosis of colon cancer cells

目的探讨T-box3基因(TBX3)对结肠癌细胞增殖、凋亡的影响,并初步探讨其机制。方法应用实时荧光定量聚合酶链反应(RT-qPCR)和蛋白印迹(Western blot)方法检测结肠癌患者及结肠癌细胞株TBX3的mRNA和蛋白表达。培养结肠癌SW620细胞株并进行干预,分为小干扰RNA(siRNA)-TBX3转染组、siRNA-对照组转染组、空白组。转染48 h后噻唑蓝(MTT)实验检测SW620细胞活性,流式细胞术检测细胞凋亡。RT-qPCR和Western blot检测各组细胞增殖、凋亡相关基因、蛋白表达。结果结肠癌患者肿瘤组织TBX3 mRNA和蛋白相对表达水平显著高于癌旁组织(3.77±0.92、0.66±0.14比0.90±0.22、0.18±0.03,t=25.02、28.21,P<0.01);结肠癌SW620细胞株TBX3 mRNA和蛋白相对表达水平显著高于HIEC(3.01±0.44、0.92±0.06比1.15±0.17、0.24±0.03),差异均有统计学意义(t=9.68、24.08,P<0.01)。siRNA-TBX3转染组SW620细胞活性明显低于siRNA-对照组、空白组(0.46±0.13、1.12±0.07、1.16±0.10,F=89.45,P<0.01);siRNA-TBX3转染组SW620细胞凋亡率明显高于siRNA-对照组、空白组[(23.09±4.59)%、(6.82±0.92)%、(7.22±1.75)%,F=31.01,P<0.01]。siRNA-TBX3转染组TBX3、增殖细胞核抗原(PCNA)、B细胞淋巴瘤(bcl-2)表达明显低于siRNA-对照组、空白组(mRNA:F=34.69、10.15、25.47,P<0.01;蛋白:F=57.56、23.10,P<0.01);siRNA-TBX3转染组bcl-2相关X蛋白(bax)、半胱天冬酶-3(Caspase-3)表达明显高于于siRNA-对照组、空白组(mRNA:F=25.17、27.79,P<0.01;蛋白:F=19.91、9.01,P<0.05);siRNA-TBX3转染组P14ARF/P53途径中P14ARF、P53蛋白表达明显低于siRNA-对照组、空白组(F=25.23、20.06,P<0.01)。结论结肠癌肿瘤组织TBX3高表达,抑制TBX3表达可抑制结肠癌细胞增殖、促进细胞凋亡,其机制可能与下调P14ARF/p53途径有关。

Objective To investigate the effect of T-box3 gene(TBX3)gene on proliferation,apoptosis of colon cancer cells,and to explore its mechanism.Methods Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the expression of TBX3 in colon cancer and cell lines.The SW620 cell line was further divided into siRNA-TBX3 transfection group and siRNA-Control transfection group,untransfected group.Methyl thiazolyl tetrazolium(MTT)assay was used to detect SW620 cell activity.Flow Cytometry was used to detect apoptosis of SW620 cells after transfected for 48 h.Expression of proliferation,apoptosis-related gene expression in each group of cells were detected via RT-qPCR and Western blotting.Results The expression of TBX3 mRNA and protein in tumor tissues of colon cancer patients was higher than that in adjacent tissues(3.77±0.92,0.66±0.14 vs.0.90±0.22,0.18±0.03,t=25.02,28.21,P<0.01).The expression of TBX3 mRNA(3.01±0.44)and protein(0.92±0.06)in colon cancer SW620 cells was higher than that in human intestinal epithelial HIEC cells(3.01±0.44,0.92±0.06 vs.1.15±0.17,0.24±0.03,t=9.68,24.08,P<0.01).The activity of SW620 cells in siRNA-TBX3 group was significantly lower than that in siRNA-control group and blank group(0.46±0.13,1.12±0.07,1.16±0.10,F=89.45,P<0.01);the activity of SW620 cells in siRNA-TBX3 group was significantly higher than that in siRNA-control group and blank group[(23.09±4.59)%,(6.82±0.92)%,(7.22±1.75)%,F=31.01,P<0.01].The expression of TBX3,proliferating cell nuclear antigen(PCNA),B-cell lymphoma/leukemia-2(Bcl-2)in siRNA-TBX3 group was significantly lower than that in siRNA-control group and blank group(mRNA:F=34.69,10.15,25.47,P<0.01;protein:F=57.56,23.10,P<0.01);and associated with X protein(Bax),caspase-3 in siRNA-TBX3 group was significantly higher than that in siRNA-control group and blank group(mRNA:F=25.17,27.79,P<0.01;protein:F=19.91,9.01,P<0.05).P14ARF and P53 protein expression of the P14ARF/P53 pathway in siRNA-TBX3 group was significantly lower than that in siRNA-control group and blank group(F=25.23,20.06,P<0.01).Conclusion TBX3 is highly expressed in colon cancer.Inhibition of TBX3 expression can inhibit proliferation,promote apoptosis of colon cancer cells.The mechanism may be related to down-regulation of P14ARF/p53 pathway.